Program Director/Principal Investigator (Last, First, Middle): Nowak, Romana A. Endometriosis is an inflammatory disease that affects 10% of women but is present in up to 70% of women with pelvic pain and 30% of women with infertility. The estimated cost of endometriosis is over $22 billion in the US alone (1), not counting associated chronic illnesses (migraine headache, irritable bowel syndrome and interstitial cystitis (2, 3). Thus it is safe to say that endometriosis is one of the costliest reproductive diseases in women not only in terms of treatment but also lost productivity and quality of life in affected patients. The most widely accepted hypothesis for development of endometriosis is retrograde menstruation that results in implantation of endometrial fragments on the visceral or peritoneal walls. Implantation of these fragments is thought to be regulated by factors secreted locally by immune cells and endometrial cells. However, the mechanisms by which endometrial cells are able to traverse the peritoneal mesothelium to establish lesions and how these lesions lead to increased inflammation and pelvic pain are not well understood. Our central hypothesis is that peritoneal mesothelial cells undergo epithelial-mesenchymal transition (EMT) in response to contact with endometrial cells and their secreted factors or with endocrine disrupting chemicals such as phthalates. This leads to a loss of peritoneal integrity allowing passage of endometrial cells into the peritoneum and induces expression of proinflammatory cytokines, chemokines and other factors that are secreted by mesothelial cells in exosomes/ microvesicles. The goal of this research proposal is to begin testing this hypothesis using an in vivo mouse model of endometriosis and an in vitro human mesothelial cell culture system to investigate the role of the peritoneal mesothelium in promoting the initiation of endometriotic lesions and how endocrine disrupters, phthalate, may affect peritoneal mesothelium and promote the development of a local inflammatory environment. We will test these hypotheses in the following two specific aims: 1) Characterize the secretome of human mesothelial cells undergoing EMT with loss of barrier integrity in an in vitro model system. We will perform small RNA sequencing and proteomic analysis of exosomes/microvesicles shed by primary human mesothelial cells undergoing EMT in an in vitro culture system. We will measure changes in trans-epithelial resistance to assess loss of barrier integrity during EMT. 2) Utilize an in vivo mouse model to investigate the process of EMT in mesothelium at sites of endometriotic lesions and the effects of phthalates on mesothelial cell EMT. We will assess changes in known EMT and inflammatory markers in early and established endometriotic lesions in mice treated with different doses of phthalates. Successful completion of these studies is expected to open up an entirely new direction in the field of endometriosis research and endocrine disrupters, providing a foundation for future studies focusing on basic mechanisms involved in inflammation and EMT and the mechanisms by which endocrine disrupters may contribute to chronic inflammation. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page